scholarly journals Identification and characterization of centromeric sequences in Xenopus laevis

2020 ◽  
Author(s):  
Owen K Smith ◽  
Charles Limouse ◽  
Kelsey A Fryer ◽  
Nicole A Teran ◽  
Kousik Sundararajan ◽  
...  
Keyword(s):  
Xenopus Laevis ◽  
Repetitive Sequences ◽  
Protein A ◽  
Repeat Sequences ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Egg Extracts ◽  
Genomic Studies

AbstractCentromeres play an essential function in cell division by specifying the site of kinetochore formation on each chromosome for mitotic spindle attachment. Centromeres are defined epigenetically by the histone H3 variant CEntromere Protein A (CENP-A). CENP-A nucleosomes maintain the centromere by designating the site for new CENP-A assembly after dilution by replication. Vertebrate centromeres assemble on tandem arrays of repetitive sequences but the function of repeat DNA in centromere formation has been challenging to dissect due to the difficulty in manipulating centromeres in cells. Xenopus laevis egg extracts assemble centromeres in vitro, providing a system for studying centromeric DNA functions. However, centromeric sequences in X. laevis have not been extensively characterized. In this study we combine CENP-A ChIP-seq with a k-mer based analysis approach to identify the X. laevis centromere repeat sequences. By in situ hybridization we show that X. laevis centromeres contain diverse repeat sequences and we map the centromere position on each X. laevis chromosome using the distribution of centromere enriched k-mers. Our identification of X. laevis centromere sequences enables previously unapproachable centromere genomic studies. Our approach should be broadly applicable for the analysis of centromere and other repetitive sequences in any organism.

scholarly journals Constitutive centromere-associated network contacts confer differential stability on CENP-A nucleosomes in vitro and in the cell

2018 ◽  
Vol 29 (6) ◽  
pp. 751-762 ◽  
Author(s):  
Shengya Cao ◽  
Keda Zhou ◽  
Zhening Zhang ◽  
Karolin Luger ◽  
Aaron F. Straight
Keyword(s):  
Protein A ◽  
Histone H3 ◽  
Intrinsic Property ◽  
Cell Cycles ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Differential Stability ◽  
The Stability

Eukaryotic centromeres are defined by the presence of nucleosomes containing the histone H3 variant, centromere protein A (CENP-A). Once incorporated at centromeres, CENP-A nucleosomes are remarkably stable, exhibiting no detectable loss or exchange over many cell cycles. It is currently unclear whether this stability is an intrinsic property of CENP-A containing chromatin or whether it arises from proteins that specifically associate with CENP-A chromatin. Two proteins, CENP-C and CENP-N, are known to bind CENP-A human nucleosomes directly. Here we test the hypothesis that CENP-C or CENP-N stabilize CENP-A nucleosomes in vitro and in living cells. We show that CENP-N stabilizes CENP-A nucleosomes alone and additively with CENP-C in vitro. However, removal of CENP-C and CENP-N from cells, or mutating CENP-A so that it no longer interacts with CENP-C or CENP-N, had no effect on centromeric CENP-A stability in vivo. Thus, the stability of CENP-A nucleosomes in chromatin does not arise solely from its interactions with CENP-C or CENP-N.

scholarly journals Propagation of centromeric chromatin requires exit from mitosis

2007 ◽  
Vol 176 (6) ◽  
pp. 795-805 ◽  
Author(s):  
Lars E.T. Jansen ◽  
Ben E. Black ◽  
Daniel R. Foltz ◽  
Don W. Cleveland
Keyword(s):  
Protein A ◽  
S Phase ◽  
Epigenetic Mark ◽  
Multiprotein Complex ◽  
Centromeric Chromatin ◽  
Microtubule Attachment ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Multiple Cell ◽  
Labeling Approach

Centromeres direct chromosomal inheritance by nucleating assembly of the kinetochore, a large multiprotein complex required for microtubule attachment during mitosis. Centromere identity in humans is epigenetically determined, with no DNA sequence either necessary or sufficient. A prime candidate for the epigenetic mark is assembly into centromeric chromatin of centromere protein A (CENP-A), a histone H3 variant found only at functional centromeres. A new covalent fluorescent pulse-chase labeling approach using SNAP tagging has now been developed and is used to demonstrate that CENP-A bound to a mature centromere is quantitatively and equally partitioned to sister centromeres generated during S phase, thereby remaining stably associated through multiple cell divisions. Loading of nascent CENP-A on the megabase domains of replicated centromere DNA is shown to require passage through mitosis but not microtubule attachment. Very surprisingly, assembly and stabilization of new CENP-A–containing nucleosomes is restricted exclusively to the subsequent G1 phase, demonstrating direct coupling between progression through mitosis and assembly/maturation of the next generation of centromeres.

scholarly journals Diaphanous formin mDia2 regulates CENP-A levels at centromeres

2016 ◽  
Vol 213 (4) ◽  
pp. 415-424 ◽  
Author(s):  
Chenshu Liu ◽  
Yinghui Mao
Keyword(s):  
Quantitative Imaging ◽  
Active Form ◽  
Protein A ◽  
Small Gtpase ◽  
Genome Replication ◽  
Rac Gtpase ◽  
Epigenetic Marker ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Recognition Protein

Centromeres of higher eukaryotes are epigenetically defined by centromere protein A (CENP-A), a centromere-specific histone H3 variant. The incorporation of new CENP-A into centromeres to maintain the epigenetic marker after genome replication in S phase occurs in G1 phase; however, how new CENP-A is loaded and stabilized remains poorly understood. Here, we identify the formin mDia2 as essential for stable replenishment of new CENP-A at centromeres. Quantitative imaging, pulse-chase analysis, and high-resolution ratiometric live-cell studies demonstrate that mDia2 and its nuclear localization are required to maintain CENP-A levels at centromeres. Depletion of mDia2 results in a prolonged centromere association of holiday junction recognition protein (HJURP), the chaperone required for CENP-A loading. A constitutively active form of mDia2 rescues the defect in new CENP-A loading caused by depletion of male germ cell Rac GTPase-activating protein (MgcRacGAP), a component of the small GTPase pathway essential for CENP-A maintenance. Thus, the formin mDia2 functions downstream of the MgcRacGAP-dependent pathway in regulating assembly of new CENP-A containing nucleosomes at centromeres.

scholarly journals Three-dimensional localization of CENP-A suggests a complex higher order structure of centromeric chromatin

2008 ◽  
Vol 183 (7) ◽  
pp. 1193-1202 ◽  
Author(s):  
Owen J. Marshall ◽  
Alan T. Marshall ◽  
K.H. Andy Choo
Keyword(s):  
Chromatin Immunoprecipitation ◽  
Protein A ◽  
Three Dimensional ◽  
Higher Order ◽  
Order Structure ◽  
Mitotic Chromosomes ◽  
Centromeric Chromatin ◽  
Centromere Protein A ◽  
Transmission Electron ◽  
Histone H3 Variant

The histone H3 variant centromere protein A (CENP-A) is central to centromere formation throughout eukaryotes. A long-standing question in centromere biology has been the organization of CENP-A at the centromere and its implications for the structure of centromeric chromatin. In this study, we describe the three-dimensional localization of CENP-A at the inner kinetochore plate through serial-section transmission electron microscopy of human mitotic chromosomes. At the kinetochores of normal centromeres and at a neocentromere, CENP-A occupies a compact domain at the inner kinetochore plate, stretching across two thirds of the length of the constriction but encompassing only one third of the constriction width and height. Within this domain, evidence of substructure is apparent. Combined with previous chromatin immunoprecipitation results (Saffery, R., H. Sumer, S. Hassan, L.H. Wong, J.M. Craig, K. Todokoro, M. Anderson, A. Stafford, and K.H.A. Choo. 2003. Mol. Cell. 12:509–516; Chueh, A.C., L.H. Wong, N. Wong, and K.H.A. Choo. 2005. Hum. Mol. Genet. 14:85–93), our data suggest that centromeric chromatin is arranged in a coiled 30-nm fiber that is itself coiled or folded to form a higher order structure.

scholarly journals Functional genomics identifies a Myb domain–containing protein family required for assembly of CENP-A chromatin

2007 ◽  
Vol 176 (6) ◽  
pp. 757-763 ◽  
Author(s):  
Paul S. Maddox ◽  
Francie Hyndman ◽  
Joost Monen ◽  
Karen Oegema ◽  
Arshad Desai
Keyword(s):  
Functional Genomics ◽  
Protein A ◽  
Common Mechanism ◽  
Centromeric Chromatin ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Close Proximity ◽  
Single Protein ◽  
Histone H3 Variant ◽  
New Protein

Nucleosomes containing the centromere-specific histone H3 variant centromere protein A (CENP-A) create the chromatin foundation for kinetochore assembly. To understand the mechanisms that selectively target CENP-A to centromeres, we took a functional genomics approach in the nematode Caenorhabditis elegans, in which failure to load CENP-A results in a signature kinetochore-null (KNL) phenotype. We identified a single protein, KNL-2, that is specifically required for CENP-A incorporation into chromatin. KNL-2 and CENP-A localize to centromeres throughout the cell cycle in an interdependent manner and coordinately direct chromosome condensation, kinetochore assembly, and chromosome segregation. The isolation of KNL-2–associated chromatin coenriched CENP-A, indicating their close proximity on DNA. KNL-2 defines a new conserved family of Myb DNA-binding domain–containing proteins. The human homologue of KNL-2 is also specifically required for CENP-A loading and kinetochore assembly but is only transiently present at centromeres after mitotic exit. These results implicate a new protein class in the assembly of centromeric chromatin and suggest that holocentric and monocentric chromosomes share a common mechanism for CENP-A loading.

scholarly journals Disulfide Bonding among μ1 Trimers in Mammalian Reovirus Outer Capsid: a Late and Reversible Step in Virion Morphogenesis

2003 ◽  
Vol 77 (9) ◽  
pp. 5389-5400 ◽  
Author(s):  
Amy L. Odegard ◽  
Kartik Chandran ◽  
Susanne Liemann ◽  
Stephen C. Harrison ◽  
Max L. Nibert
Keyword(s):  
Crystal Structure ◽  
Protein A ◽  
Cellular Membrane ◽  
Disulfide Bonding ◽  
Animal Virus ◽  
Electron Density Map ◽  
Outer Capsid

ABSTRACT We examined how a particular type of intermolecular disulfide (ds) bond is formed in the capsid of a cytoplasmically replicating nonenveloped animal virus despite the normally reducing environment inside cells. The μ1 protein, a major component of the mammalian reovirus outer capsid, has been implicated in penetration of the cellular membrane barrier during cell entry. A recent crystal structure determination supports past evidence that the basal oligomer of μ1 is a trimer and that 200 of these trimers surround the core in the fenestrated T=13 outer capsid of virions. We found in this study that the predominant forms of μ1 seen in gels after the nonreducing disruption of virions are ds-linked dimers. Cys679, near the carboxyl terminus of μ1, was shown to form this ds bond with the Cys679 residue from another μ1 subunit. The crystal structure in combination with a cryomicroscopy-derived electron density map of virions indicates that the two subunits that contribute a Cys679 residue to each ds bond must be from adjacent μ1 trimers in the outer capsid, explaining the trimer-dimer paradox. Successful in vitro assembly of the outer capsid by a nonbonding mutant of μ1 (Cys679 substituted by serine) confirmed the role of Cys679 and suggested that the ds bonds are not required for assembly. A correlation between μ1-associated ds bond formation and cell death in experiments in which virions were purified from cells at different times postinfection indicated that the ds bonds form late in infection, after virions are exposed to more oxidizing conditions than those in healthy cells. The infectivity measurements of the virions with differing levels of ds-bonded μ1 showed that these bonds are not required for infection in culture. The ds bonds in purified virions were susceptible to reduction and reformation in situ, consistent with their initial formation late in morphogenesis and suggesting that they may undergo reduction during the entry of reovirus particles into new cells.

scholarly journals Xenopus HJURP and condensin II are required for CENP-A assembly

2011 ◽  
Vol 192 (4) ◽  
pp. 569-582 ◽  
Author(s):  
Rafael Bernad ◽  
Patricia Sánchez ◽  
Teresa Rivera ◽  
Miriam Rodríguez-Corsino ◽  
Ekaterina Boyarchuk ◽  
...  
Keyword(s):  
Protein A ◽  
Human Cells ◽  
Epigenetic Mark ◽  
Temporal Specificity ◽  
Egg Extracts ◽  
Xenopus Egg ◽  
Centromeric Protein ◽  
Xenopus Egg Extracts

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction–recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

scholarly journals Identification of Xenopus CENP-A and an Associated Centromeric DNA Repeat

2005 ◽  
Vol 16 (4) ◽  
pp. 1800-1810 ◽  
Author(s):  
Nathaniel S. Edwards ◽  
Andrew W. Murray
Keyword(s):  
Dna Sequences ◽  
Cultured Cells ◽  
Protein A ◽  
Centromeric Dna ◽  
Eukaryotic Chromosome ◽  
Centromeric Repeat ◽  
Kinetochore Assembly ◽  
Centromere Protein A ◽  
Dna Repeat ◽  
Egg Extracts

Kinetochores are the proteinaceous complexes that assemble on centromeric DNA and direct eukaryotic chromosome segregation. The mechanisms by which higher eukaryotic cells define centromeres are poorly understood. Possible molecular contributors to centromere specification include the underlying DNA sequences and epigenetic factors such as binding of the centromeric histone centromere protein A (CENP-A). Frog egg extracts are an attractive system for studying centromere definition and kinetochore assembly. To facilitate such studies, we cloned a Xenopus laevis homologue of CENP-A (XCENP-A). We identified centromere-associated DNA sequences by cloning fragments of DNA that copurified with XCENP-A by chromatin immunoprecipitation. XCENP-A associates with frog centromeric repeat 1 (Fcr1), a 174-base pair repeat containing a possible CENP-B box. Southern blots of partially digested genomic DNA revealed large ordered arrays of Fcr1 in the genome. Fluorescent in situ hybridization with Fcr1 probes stained most centromeres in cultured cells. By staining lampbrush chromosomes, we specifically identified the 11 (of 18) chromosomes that stain consistently with Fcr1 probes.

scholarly journals Trans-generational inheritance of centromere identity requires the CENP-A N-terminal tail in the C. elegans maternal germ line

2020 ◽  
Author(s):  
Reinier F. Prosée ◽  
Joanna M. Wenda ◽  
Caroline Gabus ◽  
Kamila Delaney ◽  
Francoise Schwager ◽  
...  
Keyword(s):  
De Novo ◽  
Germ Line ◽  
Protein A ◽  
Histone H3 ◽  
Centromeric Chromatin ◽  
C Elegans ◽  
Centromere Function ◽  
Centromere Protein A ◽  
Histone H3 Variant ◽  
Meiosis I

AbstractCentromere protein A (CENP-A) is a histone H3 variant that defines centromeric chromatin and is essential for centromere function. In most eukaryotes CENP-A-containing chromatin is epigenetically maintained, and centromere identity is inherited from one cell cycle to the next. In the germ line of the holocentric nematode Caenorhabditis elegans, this inheritance cycle is disrupted. CENP-A is removed at the mitosis-to-meiosis transition and is established de novo on chromatin during diplotene of meiosis I. Here we show that the N-terminal tail of CENP-A is required for the de novo establishment of centromeres, but dispensable for centromere maintenance during embryogenesis. Worms homozygous for a CENP-A tail deletion maintain a functional centromere during development, but give rise to inviable offspring because they fail to re-establish centromeres in the maternal germ line. We identify the N-terminal tail of CENP-A as a critical domain for the interaction with the conserved kinetochore protein KNL-2, and argue that this interaction plays an important role in setting centromere identity in the germ line. We conclude that centromere establishment and maintenance are functionally distinct in C. elegans.

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